1. Field of the Invention
The present invention relates generally to methods and apparatus for remotely effecting spatially-selective photo-activation of one or more molecular agents and for improving the detection of the diagnostic signals thereby produced. The method taught for effecting photoactivation utilizes the special properties of non-linear optical excitation for promoting an agent from one molecular energy level to another with a high degree of spatial and molecular specificity. The special features of this method are applicable for activation of various endogenous and exogenous imaging agents, and in particular afford distinct advantages in the diagnosis of diseases in humans and animals. Specifically, use of non-linear excitation methods facilitate controlled activation of diagnostic agents in deep tissue using near infrared to infrared radiation, which is absorbed and scattered to a lesser extent than methods and radiations currently used. Combination of these non-linear excitation methods with advanced signal encoding and processing methods greatly increases sensitivity in the detection of diagnostic signals.
2. Description of the Prior Art
An urgent need exists in many fields, and especially in the medical diagnostics field, for a method that is capable of selectively controlling the remote activation of various molecular agents while producing few if any side effects resulting from the activation process. The desired improvements in activation include enhancements in spatial or temporal control over the location and depth of activation, reduction in undesirable activation of other co-located or proximal molecular agents or structures, and increased preference in the activation of desirable molecular agents over that of undesirable molecular agents. Various linear and non-linear optical methods have been developed to provide some such improvements for some such agents under very specialized conditions. However, in general the performance and applicability of these methods have been less than desired. Specifically, improved photo-activation methods are needed that may be used to selectively photo-activate a variety of molecular diagnostic agents while providing improved performance in the control of application of this photo-activation.
Application of optical radiation as a means for remotely activating molecular probes has been known for many years. Specifically, linear optical excitation methods have been used extensively as a means for achieving semi-selective activation of molecular diagnostic agents. Linear optical excitation occurs when a target agent, such as a molecular diagnostic agent, undergoes a specific photo-chemical or photo-physical process, such as fluorescent emission, upon absorption of energy provided by a single photon. These processes can in many cases be very efficient, and use of such processes is attractive for numerous applications. Unfortunately, performance of these linear methods have not always been as successful as desired. For example, there is strong evidence that ultraviolet radiation used to excite some molecular probes can produce disease in humans and animals, such as induced skin cancer, along with other undesirable side effects. Furthermore, a less than desirable penetration depth has plagued most efforts at linear optical excitation of molecular agents, primarily as a consequence of the effects of optical scatter and of absorbance of the incident probe radiation at wavelengths near the linear absorption bands of these agents. As an example, Wachter and Fisher (E. A. Wachter and W. G. Fisher, “Method and Apparatus for Evaluating Structural Weakness in Polymer Matrix Composites.” U.S. Pat. No. 5,483,338) teach of a rapid optical method capable of sensitively imaging chemical transformations in probe molecular agents; however, due to scatter and absorbance of the incident probe radiation, the method is only suitable for topical analysis. Vo-Dinh and co-workers (T. Vo-Dinh, M. Panjehpour, B. F. Overholt, C. Farris, F. P. Buckley III and R. Sneed, “In-Vivo Cancer-Diagnosis of the Esophaous Using Differential Normalized Fluorescence (Dnf) Indexes,” Lasers in Surgery and Medicine, 16 (1995) 41-47; and M. Panjehpour, B. F. Overholt, J. L. Schmidhammer, C. Farris, P. F. Buckley, and T. Vo-Dinh, “Spectroscopic Diagnosis of Esophageal Cancer: New Classification Model, Improved Measurement System,” Gastrointestinal Endoscopy, 41 (1995) 577-581) teach of the use of similar linear optical probe methods for detection of diseased tissues in humans; however, this approach is also plagued by less than desirable penetration depth and is limited to detection of superficial lesions due to scatter and absorption of the incident probe radiation. Also, because this type of excitation is linearly related to excitation power, such methods provide no effective means for limiting the location of probe excitation along the optical path. In fact, virtually all examples of the use of linear optical excitation are plagued by fundamental performance limits that are attributable to undesirable absorption and scatter of the incident optical radiation by the surrounding matrix, poor specificity in excitation of probe molecular species, and a lack of suitable physical mechanisms for precise control of the extent and depth of activation.
Various non-linear optical excitation methods have been employed in an effort to achieve specific improvements in the selectivity of photo-activation for certain applications, and to address many of the limitations posed by linear excitation methods. In fact, the non-linear process consisting of simultaneous absorption of two photons of light by a molecule to effect excitation equivalent to that resulting from absorption of a single photon having twice the energy of these two photons is very well known, as are the specific advantages of this process in terms of reduced absorption and scatter of excitation photons by the matrix, enhanced spatial control over the region of excitation, and reduced potential for photo-chemical and photo-physical damage to the sample. Excitation sources ranging from single-mode, continuous wave (CW) lasers to pulsed Q-switched lasers having peak powers in excess of 1 GW have been employed for numerous examples of two-photon excitation methods. For example, Wirth and Lytle (M. J. Wirth and F. E. Lytle, “Two-Photon Excited Molecular Fluorescence in Optically Dense Media,” Analytical Chemistry, 49 (1977) 2054-2057) teach use of non-linear optical excitation as a means for stimulating target molecules present in optically dense media; this method is shown to be useful in limiting undesirable direct interaction of the probe radiation with the media itself, and provides a means for effectively exciting target molecular agents present in strongly absorbing or scattering matrices. Improved spatial control over the active region has been further developed by Wirth (M. J. Wirth and H. O. Fatunmbi, “Very High Detectability in Two-Photon Spectroscopy,” Analytical Chemistry, 62 (1990) 973-976); specifically, Wirth teaches a method for achieving extremely high spatial selectivity in the excitation of target molecular agents using a microscopic imaging system. Similar control has been further applied by Denk et al. (W. Denk, J. P. Strickler and W. W. Webb, “Two-Photon Laser Microscopy,” U.S. Pat. No. 5,034,613) who teach of a special epi-illumination confocal laser scanning microscope utilizing non-linear laser excitation to achieve intrinsically high three-dimensional control in the photo-activation of various molecular fluorophor agents on a cellular or sub-cellular scale. This microscope is used to excite molecular fluorophor agents added to biological specimens, which constitute an optically dense medium; the special properties of non-linear two-photon excitation are utilized to substantially limit excitation and subsequent detection of the fluorescent signal thus produced to a confocal region occurring at the focus of an objective lens, thereby enhancing contrast in three-dimensional imaging by sharply controlling the depth of focus. Emitted fluorescent light is collected by the excitation objective using an epi-illumination configuration. Control of photo-excitation for generation of luminescence-based images at the cellular and subcellular level is shown in target samples mounted on a stage. Furthermore, Denk teaches that reduction in photo-induced necrosis of cells located at the focal plane is a primary benefit of this microscopy approach, based on the replacement of ultraviolet excitation radiation with less damaging near infrared excitation radiation.
In later work by Denk et al. (W. Denk, D. W. Piston and W. W. Webb, “Two-Photon Molecular Excitation in Laser-Scanning Microscopy,” in Handbook of Biological Confocal Microscopy, Second Edition, J. B. Pawley, ed., Plenum Press, New York, 1995, pp. 445-458) an external whole area detection method is taught for collection of microscopic imaging data produced from two-photon excited fluorescent tags. This method, which the authors state as being “as yet untried,” eliminates the need to collect backscattered fluorescent light using epi-illumination (see p. 452). Denk points out that this approach could be useful if the microscope objective does not transmit the emitted fluorescent wavelengths, but that it is “vulnerable to contamination from ambient room light.” In this work and in the earlier Denk patent (U.S. Pat. No. 5,034,613), no apparent method is used or anticipated for reduction of background interference from either ambient light or from scattered excitation light.
In fact, the well known low efficiency of the two-photon excitation process can translate into a very high ratio of scattered, unabsorbed excitation light to fluorescence emission. Use of various modulation methods for reduction of interference from scattered excitation light, as well as from interferences from ambient light and from other environmental and instrumental background sources, has numerous precedents. In the field of two-photon excited fluorescence, Lytle and co-workers (R. G. Freeman, D. L. Gilliland and F. E. Lytle, “Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence,” Analytical Chemistry, 62 (1990) 2216-2219; and W. G. Fisher and F. E. Lytle, “Second Harmonic Detection of Spatially Filtered Two-Photon Excited Fluorescence.” Analytical Chemistry, 65 (1993) 631-635) teach sophisticated methods for rejection of scattered laser excitation light by making use of second-harmonic detection methods: when sinusoidal modulation of the excitation light is performed at one frequency, and detection of the two-photon excited fluorescence is performed at twice that frequency (which is the second harmonic of the excitation modulation frequency), interferences from scattered excitation light are virtually eliminated. And by proper selection of the modulation frequency to avoid electronic and other noise frequencies, rejection of instrumental and environmental interferences is extremely high.
Hence, it is well known that two-photon excitation of fluorescence can be used under laboratory conditions to excite molecular fluorophors using light at approximately twice the wavelength of that used for linear single-photon excitation, and that the excitation thereby effected can improve three-dimensional spatial control over the location of excitation, can reduce interference from absorption and scatter of the excitation light in optically dense media, and can reduce collateral damage along the excitation path to living cell samples undergoing microscopic examination.
Nonetheless, while the substantial body of prior art exemplified by these cited examples clearly demonstrates many attractive features of various photo-activation methods that are applicable for diagnostic and other in vivo microscopic imaging uses, a general method for achieving selective photo-activation of one or more molecular agents with a high degree of spatial control that is capable of meeting the diverse needs of the medical diagnostic industry has not been previously taught. Specifically, practical methods for effecting such control on scales that are significant for medical diagnostic applications have not been previously taught.
It is, therefore, an object of the present invention to provide a general method for achieving selective photo-activation of one or more molecular agents with a high degree of spatial control.
It is another object of the present invention to provide such a method that is capable of meeting the diverse needs of the medical diagnostic industry.
It is another object of the present invention to provide a practical method for effecting such control on scales that are significant for medical diagnostic applications.